Continuous treatment with the D2 dopamine receptor agonist quinpirole decreases D2 dopamine receptors, D2 dopamine receptor messenger RNA and proenkephalin messenger RNA, and increases mu opioid receptors in mouse striatum

03~-4522/93 $6.00 + 0.00 Pergamon Press Ltd 0 1993 IBRO

Neuroscience Vol. 54, No. 3, pp. 669-680, 1993 Printed in Great Britain

CONTINUOUS RECEPTOR DOPAMINE MESSENGER RNA, AND

TREATMENT WITH THE D, DOPAMINE AGONIST QUINPIROLE DECREASES D, RECEPTORS, D, DOPAMINE RECEPTOR RNA AND PROENKEPHALIN MESSENGER INCREASES MU OPIOID RECEPTORS IN MOUSE STRIATUM J. F. &EN, V. J. ALOYO and B. WEISS*

Department

of Pharmacology, The Medical Coliege of Pennsylvania, 3200 Henry Avenue, Philadelphia, PA 19129, U.S.A.

Abstract-Dopamine-mediated behaviors and certain biochemical and molecular events associated with these behaviors were examined following continuous infusion of the D, dopamine agonist SKF38393 or the D, dopamine agonist quinpirole into mice for six days. SKF38393 produced a transient grooming behavior while quinpirole initially induced stereotypy, which was followed by an increased locomotor behavior. Continuous infusion of quinpirole caused a significant down-regulation of striatal D, dopamine receptors without significantly changing the density of D, receptors. This was accompanied by a decrease in the ievel of D, receptor messenger RNA in striatum as measured by Northern analysis. The down-regulation of dopamine receptors was selective for Dz dopamine receptors, since treatment with SKF38393 had no significant effects on either D, or D, dopamine receptors, nor did it alter the messenger RNAs for the D, and DZ receptors. Continuous treatment with quinpirole resulted in a si~ificant increase in striatal mu opioid receptor levels without significant changing delta opioid receptors. This treatment also induced a significant decrease in proenkephalin messenger RNA in striatum. Taken together, these results suggest that the down-regulation of D, dopamine receptor and D, receptor messenger RNA is the result of the persistent stimulation of D, receptors and that the up-regulation of mu opioid receptors may be a compensatory response to a decreased biosynthesis of enkephalin. They suggest further that the biochemical and molecular changes that take place in dopaminergic and enkephalinergic systems following continuous treatment with dopamine agonists may underlie the mechanisms by which certain dopamine-mediated behaviors occur.

haviors60,63 and a concomitant reduction in D2 dopamine receptors and D2 dopamine receptor mRNA.5A57 Conversely, long-term treatment with dopamine antagonists: denervation of dopamine terminals with 6-hydroxydopamine22,5’ or prolonged depletion of dopamine stores with reserpineg3s4’resulted in behavioral supersensitivity and an increased number of D, dopamine receptors without changing the density of D, dopamine receptors. However, chronic treatment with the dopamine antagonist haloperidol and denervation of dopaminergic nerve terminals with 6-hydroxydopamine have been shown to either increase2,5,38or have no significant effectse,2’.s2on the levels of striata! D, receptor mRNA. Since su~r~nsiti~ty of the dopamine system has been implicated in the positive symptoms of schizophrenia@ and the symptoms of tardive dyskinesia following chronic blockade of D2 dopamine receptors,‘“’ selective down-regulation of the dopamine system may suggest a novel strategy for treating disorders associated with dopaminergic supersensitivity. However, relatively little is known about the specific molecular events responsible for dopamine agonist-induced subsensitivity of the dopamine system and even less is known about the molecular

Central dopaminergic neurons contribute profoundly to a variety of behavioral and motor functions. Abnormal activity of the dopaminergic system has been well documented in the pathophysiology of schizophrenia, ‘3~ ParkinsonismM and tardive dyskinesia.3 However, the specific molecular mechanisms underlying these disorders are still unclear. Studies from several laboratories have provided strong evidence that the dopaminergic system is remarkably plastic in adapting to changes in neuronal inputs. For example, continuous treatment with dopaminergic agonists caused a down-regulation of dop~ine-mediated behaviors.6~s9 This down-regulation of dopamine-mediated behavior was selective in that ~ntinuous treatment with D, agonists caused a don-regulation of D,- but not D,mediated behaviors.” Similarly, continuous treatment with D, agonists resulted in a reduction of D2-mediated be*To whom correspondence should be addressed. DAMGO, (o-Ala*,NMe-Phe4,GlyS-01) enkephalin; DPDPE, (o-Pen*@-Pen’) enkephalin; SKF 38393, (2,3,4,5,-tetrahydro-7,&dihydroxy-I-phenyl-lH-3 benzazeuine HCl): SCH23390. IR(+)-8-chloro-2,3.4.5,tetrahy~ro-3-meihyl-5-phenyi-iH:3-benzazepin~-;-~li; SSC, standard saline citrate.

Abbreviations:

669

670

.I 1”. (‘HE3 <‘I rd.

events involved in the interaction between the dopamine system and other systems that appear to be involved in dopaminergic behavior, such as the chohnergic, GABAergic and peptidergic systems. Accordingly. in this study, we have concentrated on the complex functional interactions that exist between the dopamine and enkephalin systems. The corpus striatum is a likely site for an interaction between the dopamine and enkephahn systems because it contains particularly high levels of dopamine and enkephahn, as well as their receptors.‘3,24,3’ In striatum, enkephalin mRNA is co-localized to the medium spiny neurons containing Dz receptor mRNA.‘9~28Furthermore, denervation of the nigrostriatal system’2~20.62 or chronic treatment with dopamine antagonists34,“5 induce postsynaptic dopamine supersensitivity. as well as increases in striatal enkephalin content and proenkephalin mRNA. Finally, enkephalin and its analogs, such as the mu receptor agonist (~-Alaz,NMe”Phe4,G~y5-ol)enkephalin (DAMGO) or the delta receptor agonist (D-Pen’,D-Pen’)enkephalin (DPDPE), have been shown to induce changes in dopamine release,‘“.46 dopamine receptor density’* and dopamine-mediated behaviors.26.27,30.47.50

successful cloning of the genes for the dopamine receptors” and proenkephahn” offered us an opportunity to study the modulation of the dopamine and enkephalin systems at the level of gene expression. In the present study, we examined the effects of continuous infusion of the D, dopamine agonist SKF38393 and the Dz dopamine agonist quinpirole on the modulation of dopamine and opioid receptors, and examined the effects of these agonists on the mRNAs for the dopamine receptors and proenkephalin in mouse striatum. We attempted to correlate these biochemical and molecular events with certain dopamine-mediated behaviors. Recent

EXPERIMENTAL

PROCEDURES

Animal and treatment

Male Swiss Webster mice (34-37 g) were purchased from Ace Animal Inc. (Boyertown, PA). The animals were housed in plastic cages and provided free access to food and water. The animals were maintained in temperature- and humiditycontrolled rooms with a 12-h light-dark cycle. Mice were continuously infused with vehicle (12.5% ascorbic acid and 50% dimethylsulfoxide), SKF38393 (RBI, 16 ~mol/kg/h) or quinpirole (RBI, 8 ~moi~g/h) by implanting Alzet miniosmotic pumps as described previously.59.63Drug-induced behavioral changes were evaluated (see below) during a six-day period of infusion. The animals were then killed by decapitation” The brains were either dissected for Northern analysis or frozen whole and sectioned (10pm) for receptor autoradiography and in situ hybridization histochemistry. Evaluation of behavior

The rating scale system for evaluating behavior was modified from that of Sturgeon et 01.~’The behaviors were evaluated in a single-blind fashion by a trained observer at various times after the implantation. For each observation period, the mice were rated for 20-s periods at 5 min

intervals for a total of 6Omtn. The animals were tatcd tick locomotion. grooming and stereotypy at various times after the implantation, as described previously.“’ The m;tximal score attainable using this system is 36. Receptor autoradiogruph)

D, and D? dopamine receptors were detected usmg (3H]SCH23390 and [3H]spiperone, respectively, according to procedures described previously.‘-” BrietIy, striatal sections were prewashed with cold 50 mM Tris buffer (pH 7.7) and then incubated at room temperature for 6Omin with 2.0nM [iH]SCH23390 (NEN,85.6~i~mmol) for the D, dopamine receptor or 6,OnM [3H]spiperone (NEN. 24.9 Ci/mmol) and ketanserin (40 nM) for the D2 dopamine receptor. The sections were washed in ice-cold Tris buffer three times for 1min each. To define non-specific binding for the D, and I$, dopamine receptors, 2.0 PM SCH23390 or I.O,nM sulpiride. respectively. were co-incubated in adjacent sections. Mu and delta opioid receptor binding was perfomled as described earlier. ’ ” 54For mu opioid receptors, the sections were incubated with 5nM [‘HIDAMGO (NEN,47.8 Ci: mmol) in 50 mM Tris buffer (pH 7.4) at room temperature for 60 min. For delta opioid receptors, the sections were incubated with 0.3 nM [jH] DPDPE(C1) (NEN.46.0 Gil mmol), in 50mM Tris buffer, containing 5mM Mg” LII room temperature for 2.5 h. The sections were then washed three times for 1mitt each in ice-cold Tris buffer. Adjacent sections were incubated in the presence ofnaioxone (IO y M) to define non-specific binding. In vitro opioid receptor hindmg o.v.sa~:s Opioid receptor binding assays were performed using washed membrane preparations from whole mouse brain (excluding cerebellum).” Briefly, the brains were homogenized in 20 vols of SOmM Tris buffer (pH 7.4) and centrifuged at 48,000 x g for 20 min. The pellet was resuspended in Tris buffer, incubated at 25-C for 30 min, recentrifuged and resuspended in the buffer. The membrane preparations were incubated at 25’C for 1.5 and 3.5 h with 0.3 nM [ZH]DAMGO or 0. I nM ~~H]DPDPE(~l), respectively. in the absence or presence of various con~ntraiions of SKF38393 and auinnirole, The mu ooioid agonist dermorphin (70 nM) and ihe delta opioid agonist heltorphin (240 nM) were used to define non-specific binding of the mu and delta opioid receptors, respectiveiy.‘s tcsO values were determined by a non-linear least squares regression method using Ligand computer programs.” Oli~odeoz~~nucleoti(ieprobes

The two ohgodeoxynucleotide probes for the Dz receptor [common and insert probes), which were characterized in previous studies, 8,ywere used in these experiments. These probes are specific for detecting total D, receptor mRNA (common probe) or the long isoform of the D, receptor mRNA (D,,, insert probe). The olig~eoxynucleotide probe that we designed for the D, receptor mRNA (5’-GAC-ATCGGT-GT~-ATA-GTC-.CA~-TAT-GAC-CGA-TAAGGC-3’) is comulementary to a oortion of the mRNA coding the aminoacid sequence 419430 of the D, dopamine receptor.M The ohgodeoxynucleotide probe for the proenkephalin mRNA was the same as that described previously. Q The probes were synthesized by Biosynthesis Laboratories (Denton, TX) and radiolabeled at the 3’-end with alpha [ -‘S]dATP (NEN,l400 Ci/mmol) for in situ hybridization or [ ‘*PI-dATP (NEN.3000 Ci/mmol) for Northern analysis, using terminal deoxynucleotidyl transferase as described by Lewis et uI.‘~ Northern ana~~~sisand in situ h.yhridization histochemistry

Northern analysis was carried out by the method described by Davis et at.‘” Total RNA was extracted by lithium chloride--guanidinium thi~yanate-phenol~hloroform extraction, as described by ~hromcz~ski and

671

Quinpirole modufates striatal dopamine and enkephalin RESULTS

Sacchi.” RNA content was quantified by measuring the absorbance at 260 and 280 nm. The RNA was fractionated on a 1% agarose gel, containing 0.66 M formaldehyde, and transferred onto nitroceihdose membranes. The membrane was prehybridized for 2 h at room temperature in 4 x SSC (I x SSC = 0.15 M sodium chtoride/0.025 M sodium citrate), $0 x Denbardt’s solution and ~~~~rnl salmon sperm DNA, and then hybridized with about I x 106c.p.m./ ml (about 0.13nM) of 3ZP-fab&d otigodeoxynucleotide probe in the same buffer. The blot was washed three times at 55°C and once at 65°C (30min each) with 2 x ssc. In situ hybridization histochemistry was carried out according to the procedures described previously.Bs9 Briefly, tissue sections were postfixed in 4% ~rafo~aldeh~e for 5 min. After brief rinses in 0. I M phosphate-buffed saline and 2 x SSC, each section was overlaid with Mopi of hybridization buffer, containing 0.4 nM “S-labeled oligodeoxynucleotide probes. The &tions were incubated- at 37°C overnight, then washed in 1 x SSC for 2 h at room temperature and washed at a final stringency of 0.5 x SSC at 48°C.

Behavioral effects of continuously infusing quinpirole and SKF38393

Consistent with the results of earlier studies, continuous treatment with SKF38393 produced a transient grooming behavior, whereas continuous treatment with quinpirole induced an initial stereotypy which was followed by a locomotor behaviaP5,63 (Fig. 1). Neither vehicle- nor SKF38393-treated animals showed any significant stereotyped or locomotor behaviors. SKF38393 produced significant grooming within 1 h after beginning the infusion (Fig 1). This grooming behavior returned to control levels at 4 h. At no time did quinpirole produce significant grooming behavior. E$ects of ~o~ti~uous~y infusing quinpiroIe and SW38393 on D, and D2 dripamine receptors and mu and delta opioid receptors

Image and statisticalanalyses The hybridization signals of the Northern blot and in hybridization studies were detected by film automdiography. X-ray films from receptor autoradiography, Northern blot and in situ hybridization were subjected to densitometric analysis using a Drexel University hnage Analysis System (DFJMAS) as described previously.8*g For the receptor autoradio~aphi~ studies, sin&e statistical comparisons of the drug-tested group to vehicle group were performed using an independent Student’s t-test. To minimize the experimental variation in the in situ hybridization studies, sections from a pair of mouse brains from vehicle- and drug-tested animals were mounted on the same slides and processed at the same time. The data are expressed as a percentage of the values from vehicle-treated animals. A one-group r-test was used to determine the statistical significance of differences. situ

Receptor autorad~ographic analyses of dopamine and opioid receptors in corona1 sections through mouse striatum show specific labeling of D, (i3H]SCH23390) and Dz ( [ ‘Hlspiperone) dopamine receptors, respectively, in s~~~tu~ and olfactory tubercle (Fig. 2). Continuous infusion with quinpirole decreased Dz dopamine receptors in striatum but had no obvious effect on D, dopamine receptors (Fig. 2). Quantitative analysis of autoradio~ams from six similar experiments showed that quinpirole reduced the D, dopamine receptor density by I?% without significantly changing D, dopamine receptor density

T

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STEREOTYPY

-

LocxWXIQN(CIU1N)

‘iA- -

OROOMtNG

(QUIN)

(SKF)

1000

HOURS

AFTER

DEPLANING

AGONIST

Fig. 1. Effects of continuous infusion of dopaxnine agonists on dopamine-mediated behaviors in mice. Mice were ~on~nuo~~y administrated the D, agonist SKF38393 (16 ~mol~~h~, the T&agonist quinpirote (8~mol~g/h) or vehicle (50% dimethyisu~foxide and 12.5% ascorbic acid) via osmotic minipumps. Stereotypy, locomotion and grooming behaviors were determined during a six-day period of infusion, as described in Experimental Procedures. SKF38393 produced a significant grooming behavior whereas quinpirole induced significant stereotypic and locomotor behaviors. Each point represents the mean value from IO animals. Vertical bars indicate the standard errors.

672

J. F.

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VEHICLE

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SKF38393

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QUINPtROLE

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Fig. 2. Effects of continuous infusion of dopamine agonists on dopamine and opioid receptors in mouse striatum. Mice were continuously infused with SKF38393, quinpirole or vehicle as described in Experimental Procedures. The D, and D, dopamine receptors were detected by ‘H-labelled SCH23390 and spiperone, respectively, and the mu and delta opioid receptors by [‘HJDAMGO and [‘H]DPDPE(CI). respectively, using receptor autoradiography on brain sections through the mouse striatum. The figure shows that SKF38393 had no significant effect on the binding of any of the ligands studied. By contrast, quinpiroie decreased D, dopamine receptor binding without significantly changing D, dopamine receptor binding. Quinpirole treatment also significantly increased the levels of mu opioid receptors in striatum without changing the delta opioid receptor ST, corpus striatum; OT, olfactory tubercle

(Fig. 3A). By contrast, continuous treatment with SKF38393 produced little effect on either D, or Dz dopamine receptor levels (Figs 2, 3A). Analysis of the mu and delta opioid receptors (Fig. 2) showed that mu opioid receptors were found in dense patches throughout the striatum, with little labeling in olfactory tubercle. By contrast, the labeling of delta opioid receptors was homogeneously distributed in striatum, with weak labeling in olfactory tubercle. Continuous infusion of quinpirole in-

duced a significant (60%; P ~0.05) increase in mu opioid receptors in striatum, but no significant change in delta opioid receptors (Fig. 3B). The increase in striatal mu opioid receptor level was specific for D, receptor activation, since no significant change of the mu opioid receptor was observed following SKF38393 infusion (Figs. 2, 3B). The effects of various concentrations of SKF38393 or quinpirole added in vitro on the mu and delta opioid receptors were determined using washed brain

613

Quinpirole modulates striatal dopamine and enkephalin preparations. SKF38393 inhibited both [ ‘H]DAMGO and [ 3H]DPDPE(Cl) binding, with lcsO values of 1.0 and 8 PM, respectively (Fig. 4). By contrast, quinpirole failed to inhibit [ ‘H]DAMGO or [ ‘H]DPDPE(Cl) binding in mouse brain until concentrations were at least 10 times higher than that likely to be attained in vivo. The ICY values for quinpirole in inhibiting [ ‘H]DAMGO and [ ‘H]DPDPE(Cl) binding were 100 and 4QOOPM, respectively (Fig. 4). To eliminate possible effects of residual drugs on the measurement of dopamine and opioid receptor binding, three groups of animals (n = 4 per group) were acutely injected (i.p.) with either SKF38393 (16 pmol/kg), quinpirole (8 pmol/kg) or vehicle (50% dimethylsulfoxide and 12.5% ascorbic acid). The animals were killed 40min after the injections (the time at which peak behaviors were produced) and processed for receptor autoradiography. The receptor densities in vehicle- and drug-treated groups are shown in Table 1. As may be seen, acute injections of either SKF38393 or quinpirole, at the doses used, failed to produce any significant effects on the binding of the ligands to D, or D, dopamine receptors or to mu or delta opioid receptors in mouse striatum.

membrane

Effects

of

continuously

infusing

quinpirole

and

SKF38393 on the mRNAs for the dopamine receptors and proenkephalin

The specificity of the oligonucleotide probes for the D, receptor mRNA and proenkephalin mRNA has been characterized in previous studies.‘.’ The specificity of the oligodeoxynucleotide probe for the D, receptor mRNA was confirmed as follow: (i) North-

em analysis demonstrated a single band at the expected molecular size (4.6 kb) for the D, receptor mRNA (not shown); (ii) in situ hybridization histochemistry revealed that the D, receptor mRNA was specifically labeled in mouse striatum and olfactory tubercle (see Fig. 7); (iii) the hybridization signal could be displaced by preincubation with 100 times excess unlabeled oligodeoxynucleotide probe for the D, receptor mRNA but not by the probe for the Dz receptor mRNA (not shown). Northern analysis revealed that continuous infusion with quinpirole selectively down-regulated the mRNAs for the D, dopamine receptors (by common probe) and proenkephalin in the striatum by 18% and 36%, respectively (Figs 5, 6). The hybridization signal of the D, receptor mRNA in the Northern blot was too weak to be analysed quantitatively by the DUMAS image analysis system. Studies using in situ hybridization histochemistry showed that continuous infusion of quinpirole (Figs 7, 8) significantly reduced the levels of proenkephalin mRNA. However, the decrease in D, receptor mRNA detected by either the common probe (Figs 7, 8) or insert probe (not shown) failed to reach statistical significance. Continuous treatment with SKF38393 failed to produce any significant changes in the mRNAs for the D, dopamine receptor or proenkephalin in mouse striatum when measured by either Northern blot (Figs 5, 6) or by in situ hybridization histochemistry (Figs 7, 8). Furthermore, no significant change in the D, receptor mRNA was observed by in situ hybridization following treatment with either SKF38393 or quinpirole (Figs 7, 8).

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VEHICLE

n

DUINPIROLE

n

w38393

3H-SPIPERONE

B

*

3H-DAMQO

3H-DPDPE(CL)

Fig. 3. Quantitative analysis of the effectsof quinpirole and SKF 38393 on D, and D, dopamine receptors and mu and delta receptors in mouse striatum. Drug treatments and receptor autoradiographic analysis were performed as described in Fig. 1. The autoradiograms were quantified densitometrically using the DUMAS image analyses system. Receptor levels were expressed as fmol/mg tissue after calibration with tritium standards. Each value is the mean of six mice. lP ~0.05 compared with vehicle-treated animals.

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Fig. 4. Effects of SKF38393 and quinpirole added in vitro on [‘HJDAMGO and [3H]DPDPE(Cl)binding in mouse brain. The specific binding of [ ‘HIDAMGO (for mu opioid receptors; A) and [ )H]DPDPE(Cl) (for delta opioid receptors; B) was measured in washed membranes prepared from mouse brain as described in Experimental Procedures. Binding was determined in the absence and presence of various concentrations of SKF38393 and quinpirole.

DISCUSSION

Continuous treatment with quinpirole decreases striatal D, dopamine receptors and D, receptor mRNA

Earlier studies showed that certain dopaminemediated behaviors can be selectively reduced by Table 1. Effect of acute

injections of SKF38393 quinpirole on the in vifro binding of various dopamine opioid ligands to mouse striatal membranes Receptor (fmol/mg Ligand

D, ([3H]SCH23390) D, ([3H]Spiperone) p([‘H]DAMGO) G([3H]DPDPE(Cl))

Vehicle 655 k 21 263 & 9 165+8 115+6

and and

density tissue)

SKF38393 674 + 12 269 f 10 149 * 17 115*5

Quinpirole 674 _+ 16 271 k 16 155+6 118k3

Rats were acutely injected with either SKF38393 (16 pmol/kg, i.p.), quinpirole (8 pmol/kg, i.p.) or vehicle. The animals were killed 40 min after the injection and processed for receptor autoradiography. Receptor densities are expressed as fmol/mg tissue after calibration with tritium standards. Acute injections of either SKF38393 or quinpirole failed to produce any significant effects on the binding of the ligands to D, and D, dopamine receptors or to mu or delta opioid receptors in mouse striatum. Each value is the mean f SE. of four experiments.

continuously infusing specific dopamine agonists into mice.55.5”~~63 This is seen both in normal mice5’x6’and in mice rendered supersensitive by injecting 6-hydroxydopamine unilaterally into the striatum.5’.60 The present studies, showing that continuous treatment of normo-sensitive mice with the D, dopamine agonist SKF38393 and the D, dopamine agonist quinpirole down-regulates grooming and stereotyped behaviors, respectively, support those findings. To maximize the down-regulation of dopaminergic responses, in the present studies we used higher doses of quinpirole than those used earlier; accordingly, quinpirole produced more dramatic changes in stereotyped behaviors. The results using normal mice are different, in some respects, from those obtained in supersensitive mice lesioned with 6-hydroxydopamine. In 6-hydroxydopamine-lesioned mice, behavioral desensitization occurred to a greater degree and was produced more rapidly following continuous treatment with SKF38393 than with quinpirole.58~60Different rates at which behaviors are inhibited may therefore be explained not only by the type and dose of desensitizing agents used and the types of receptors involved, but also by the model system studied, i.e. whether normal or supersensitive behaviors are examined.

675

Quinpirole modulates striatal dopamine and enkephalin

21,8kb-

4D2R

1.4kb-

-ENK

Fig. 5. Effects of continuous treatment with SKF38393 and quinpirole on the mRNAs for the D, dopamine receptors and proenkephalin in mouse striatum as detected by Northern blot. Following continuous infusion of SKF38393 (SKF). auinnirole (QUIN) or vehicle (VEH) for six davs. total RNA was isolated from mouse striatum’and Northern blot analysis performed as described in Experimental Procedures. The figure shows bands corresponding to D, receptor mRNA (D,R; 2.8 kb) and proenkephalin mRNA (ENK; 1.4 kb). The 18s band represents the relative amounts of total RNA loaded onto each well.

Using receptor binding assays of striatal membranes, it was suggested that the down-regulation of D,-mediated behaviors was due to a reduction in D, dopamine receptors.@’ The present results support these findings and show further that the decrease in D2 dopamine receptors can be evidenced using receptor autoradiographic analysis of striatal sections. They also showed that the down-regulation of D, dopamine receptors by quinpirole was due to its chronic actions rather than to any acute, direct effects, or to any effects the residual drug may have on these receptors. In addition, the results show that the D, dopamine agonist quinpirole is selective in that it failed to alter D, dopamine receptors in striatum, results that are also consistent with previous findings using striatal membranes.@’ The down-regulation of D2 dopamine receptors induced by continuous infusion of quinpirole was accompanied by a decrease in D2 receptor mRNA in striatum detected by Northern analysis. This is in agreement with results of previous studies using quinpirole” and with those showing that dopaminergic denervation or chronic treatment with dopamine antagonists increase D, receptor mRNA.5,28.38 However, other investigators,8,2’~52using similar treatments, failed to demonstrate significant effects on striatal D, receptor mRNA. This apparent inconsistency may be explained by the complex changes that take place in D, receptor mRNA over time.2 Apparent discrepancies in the literature might also be explained by the differences in sensitivity between in situ hybridization histochemistry and Northern analysis, and by the fact that the changes that take place in D, receptor mRNA following dopaminergic perturbations are quite subtle. Consistent with this premise, Gerfen et al.,19 using in situ hybridization histochemistry, recently reported that 6-hydroxy-

VEHICLE

D2R

mRNA

ENK

mRNA

Fig. 6. Quantitative analysis of the effects of quinpirole and SKF38393 on the mRNAs for the D, dopamine receptor (D2R) and proenkephalin (ENK) as detected by Northern blot. Drug treatments and Northern blot analysis were performed as described in the legend to Fig 5. The mRNA levels were quantified densitometrically using the DUMAS image analysis system. Each value obtained from drug-treated animals is corrected for the amount loaded onto the gel and is expressed as a percentage of paired control (vehicle-treated) animals. Each value is the mean from eight experiments for D, receptor mRNA and five experiments for proenkephalin mRNA. *P co.05compared with vehicle-treated mice.

dopamine lesions resulted in a small (about 5%) increase in striatal D, dopamine receptor mRNA and that continuous treatment with quinpirole completely reversed this increase. Taken together, these results suggest that the quinpiroie-induced down-regulation of the D, dopamine receptors in striatum may be explained, at least in part, by decreases in D, receptor mRNA. In contrast to the results obtained with quinpirole, continuous treatment with SKF38393 failed to significantly change the D, and D, dopamine receptors or the mRNAs for the D, and D, dopamine receptors and proenkephalin in striatum. These results are in agreement with the observations that chronic treatment with neuroleptics or dopaminergic denervation with 6-hydroxydopamine cause more consistent changes in D2 dopamine receptors than in D, dopamine receptors. 3.22The biochemical and molecular basis for the down-regulation of D,-mediated grooming behavior is, therefore, stifi unclear. Nevertheless. these results suggest that D, and Dz dopamine recep-

VEHlCLE

tors are regulated by different mechanisms and that D, dopamine receptors are more susceptible to pharmacological modulation than D, dopamine receptors. Continuous treatment with quinpirole decreuses proenkephalin mRNA and increases mu opioid receptors

Continuous infusion of quinpirole decreased striatal proenkephaiin mRNA in mouse striatum. This is consistent with the results of Gerfen et ai., “J who showed that continuous treatment (but not intermittent injection) with quinpirole reversed the increased level of striatal proenkephalin mRNA induced by 6-hydroxydopamine lesions. An increase in proenkephalin mRNA would be expected to increase enkephalin peptides. Since enkephalin has a relatively high affinity for both mu and delta opioid receptors,14 it was of interest to examine the effects of dopamine agonists on mu and delta opioid receptors. Continuous treatment with quinpirole selectively increased mu but not delta opioid receptors in stria-

SKF38393

Fig. 7. Effects of continuous infusion of quinpirole and SKF38393 on the mRNAs for the D,and D, dopamine receptors (D,R and D,R) and proenkephalin (FINK) as detected by in situ hybridization histochemistry. Mice were administrated SKF38393, quinpirole or vehicle for six days and in situ hyb~di~tion of coronal sections of mouse brain (a&the level of the striatum) was performed as described in Experimental Procedures. The hybridization signals were analysed by film autoradio~raphy. ST, corpus striatum: OT. olfactory tubercle.

671

Quinpirole modulates striatal dopamine and enkephalin

c F0

nae 0’

q

VEHCLE

n

QUINPIROLE

n -w

4a

DIR

mRNA

DZR

mRNA

ENK

mRNA

Fig. 8. Densitometric analysis of effects of quinpirole and SKF38393 on the mRNAs for the D, and D, dopamine receptors (DIR and D2R) and proenkephalin (ENK) in mouse striatum as detected by in situ hvbridization histochemistrv. The mRNAs for the D, and D, dopamine receptors and proenkephalin were detected by in situ hybridization histochemistry in mouse stiiatnm treated with SKF38393, quinpirole or vehicle. The hybridization signal was detected by film autoradiography and quantified densitometrically using the DUMAS image analysis system. The optical density values of drug-treated animals were normalized to values obtained from paired control animals and were expressed as a percentage of the control optical density. Each value is the mean of five animals. *P co.05compared with control (vehicle-treated) mice.

turn. Studies of mouse brain using various concentrations of [ HjDAMGO and [ ‘H]DPDPE(Cl) indicated that these ligands had Kd values of 0.5 and 0.07 mM, respectively. Since we used concentrations of 5 nM for [ ‘H]DAMGO and 0.3 nM for [)H]DPDPE(Cl), the concentrations used in the present

studies were likely to saturate the receptors. These results suggest, therefore, that quinpirole increased the density of mu opioid receptors rather than changed the affinity of the receptors for the ligand. The up-regulation of the mu opioid receptor was specific for the activation of D2 dopamine receptors, since continuous treatment with SKF38393 had little effect on mu or delta opioid receptors. This is in agreement with reports showing that proenkephalin mRNA is specifically co-localized to striatal neurons expressing D, receptor mRNA19~Z8and that dopaminergic modulation of enkephalin and proenkephalin mRNA is through an action on D, dopamine receptors.20~36 The mechanism underlying the up-regulation of mu opioid receptors by quinpirole may involve a direct action of quinpirole on mu but not delta opioid receptor sites. It has been shown that several dopamine antagonists inhibit the binding of a number of opioid receptor ligands in vitro.4 The results showing that acute treatment with quinpirole did not have any significant effects on mu opioid receptors in striatum suggest that quinpirole did not have a direct action on mu opioid receptors sites. We examined this possibility further by carrying out in vitro competitive inhibition studies using a washed brain membrane preparation. Quinpirole failed to inhibit mu or delta

opioid receptors at concentrations likely to be attained in uivo. In contrast, SKF38393 inhibited both mu and delta opioid binding at concentrations that might be attained in vivo. The relative potency of

these dopamine agonists to inhibit mu and delta binding in vitro is in contrast to their in vivo effects on mu and delta opioid receptor levels. Treatment with quinpirole in vivo significantly increased mu but not delta opioid receptors, whereas SKF38393 had little effect on mu or delta opioid receptors. These results argued against quinpirole affecting mu opioid receptor levels through a direct action on either the mu or delta opioid receptors. Alternatively, up-regulation of mu opioid receptors by quinpirole may be a compensatory response to dopaminergic inhibition of enkephalin biosynthesis. Consistent with this hypothesis, we found that quinpirole decreased proenkephalin mRNA in mouse striatum. This is also in agreement with previous findings that chronic treatment with dopamine antagonists increased the levels of both proenkephalin mRNA and enkephalin peptide20~34*3s and decreased the density of mu opioid receptors in striatum.48 Since enkephalin has relatively similar affinities for both mu and delta opioid receptors, the mechanism responsible for the selective up-regulation of mu opioid receptors needs to be clarified. That the up-regulation of mu opioid receptors plays a role in certain dopamine-mediated behaviors is supported by the evidence that activation of opioid receptors increases locomotor behaviors.27z39Since the mu opioid receptor was selectively modulated by quinpirole, we speculate that this receptor may be involved in quinpirole-induced locomotor behaviors. Activation of D, dopamine receptors or mu opioid receptors inhibits dopamine-sensitive adenylate cyclase.42,tis53Therefore, up-regulation of mu opioid receptors may exert synergistic effects with D2 agonists on the inhibition of cyclic AMP production, and further strengthens the disinhibitory effects of the output from the basal ganglia, resulting in an increase

in locomotor activity. The evidence that mu opioid receptors are changed under several other pharmacologic treatments and pathological conditions, all of which result in abnormal motor behaviors.‘7.2r.“9 supports the suggestion of a possible involvement of mu opioid receptors in locomotor behaviors.

CONCIAJSIONS

Continuously infusing the D, agonist SKF38393 produced a transient grooming behavior, whereas continuously infusing the D2 agonist quinpirole induced an initial, transient stereotypy and a laterdeveloping locomotor behavior. Continuous administration of quinpirole produced a down-regulation of

Dz dopamine receptors and a concomitant decrcxc in D2 receptor mRNA, without signiticantI> changing the density of D, dopamine receptors OJ- the le\d 01‘ D, receptor mRNA in mouse striatum. Quinpirolc treatment also significantly decreased the !evels of striatal proenkephalin mRNA and increased striatal mu but not delta opioid receptors. These biochemic,ii and molecular changes that take place in the dopamine and enkephalin systems may help explain certain

dopamine-mediated

behaviors.

Ackno,r,[ed~rments-This work was supported, m part. hy Grants MH 42148 (B.W. and J.F.C.) and MH I6841 (V.J.A.) awarded by the National Institute of Mental Health. We thank Dr Ed Gracely for consultations concerning the statistical analyses.

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